Method to treat multiple sclerosis with GP39-specific antibodies

ABSTRACT

Method for the treatment of multiple sclerosis and other T cell mediated autoimmune disorders is described. The method involves administering to a subject a therapeutically effective amount of an antagonist of a receptor on a surface of a T cell which mediates contact dependent helper effector functions, for example, an anti-gp39 antibody.

This application is a continuation of application Ser. No. 08/481,735,filed Jun. 7, 1995, now U.S. Pat. No. 5,833,987.

GOVERNMENT FUNDING

The work leading to this invention was supported by one or more grantsfrom the U.S. government. The government may have rights in theinvention.

BACKGROUND OF THE INVENTION

Autoimmune diseases are characterized by attack of the immune system ofan individual against its own tissues. Autoimmune diseases usuallyresult from breakdown of tolerance of the immune system to its ownantigens. The specific antigens recognized by the immune system in thevarious autoimmune diseases can be present systematically or they can beorgan specific. For example, systemic lupus erthematosus (SLE) ischaracterized by the presence of autoantibodies to DNA,ribonucleoproteins, histones, and other molecules that are not organspecific. Other autoimmune diseases are characterized by the destructionof mostly one organ. Such autoimmune diseases include type I diabetes,in which the insulin producing β cells of the islets of Langerhans inthe pancreas are destroyed.

In some autoimmune diseases, tissue destruction occurs primarily as aresult of the production of high levels of autoantibodies. Such diseasesinclude rheumatoid arthritis, characterized by destruction of the jointcartilage and inflammation of the synovium. Patients with rheumatoidarthritis have an accumulation of immune complexes in their joints whichare formed by association of autoantibodies against the Fc portion ofIgG and IgG molecules. These immune complexes activate the complementcascade which results in tissue damage. Myasthenia gravis, a disease ofprogressive muscle weakness, is caused by the production ofautoantibodies reactive to acetylcholine receptors in the motor endplates of neuromuscular junctions.

In other autoimmune diseases, tissue destruction does not appear to beprimarily mediated by production of autoantibodies, but rather byauto-reactive T lymphocytes. For example, experimental allergicencephalomyelitis (EAE), an animal model for multiple sclerosis, andcharacterized by demyelination in the brain and the spinal cord, can beinduced in naive animals by transfer of CD4+ T cells from diseasedanimals. Thus, it is generally considered that EAE represents a T cellmediated autoimmune disease, rather than a B cell mediated autoimmunedisease (Ben-Nun, A. et al. (1981) Eur. J. Immunol., 11, 195).

Multiple sclerosis (MS) is a common demyclinating disease of the brainand spinal cord. It is a progressive disease that is characterized byremissions and exacerbations of neurologic dysfunction affectingdifferent regions of the central nervous system. The symptoms of thedisease result from a focus of inflammatory demyelination, which laterforms a scar, appearing as a “plaque” in the white matter of the brain,brain stem or spinal cord. Presently, there is no definitive diagnostictest available for MS and diagnoses and treatment regimes are beingformulated based on such factors as the extent of a patient's symptomsand/or the age of the patient at the time of onset of the exacerbationsof neurologic dysfunction.

Patients having MS typically have been treated with steroids with a goalof either sending the patient into remission or slowing the progressionof the disease in the patient. Other drugs have been used to treatparticular symptoms of the disease, e.g. muscle relaxants. Recentdevelopments in treatments available for MS include the administrationof beta-interferon. Beta interferon has shown some promise for slowingthe progression of the disease. However, effective treatments for MS arestill needed.

SUMMARY OF THE INVENTION

This invention pertains to methods for treating (therapeutically orprophylactically) a T cell mediated autoimmune disorder, such asmultiple sclerosis. The method comprises administering to the subject atherapeutically or prophylactically effective amount of an antagonist ofa receptor on a surface of a T cell which mediates contact dependenthelper effector functions. In a preferred embodiment the antagonistadministered is an antibody or fragment thereof which specifically bindsto the T cell receptor gp39.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of DAS units measured daily in miceinjected at day 0 with 75 μg (Panel A) or 300 μg (Panel B) ofPLP-peptide with an anti-gp39 antibody (black bars) or with PBS (greybars) showing that anti-gp39 administration prevents the development ofexperimental allergic encephalomyelitis EAE.

FIG. 2 is a graphical representation of the percentage suppression ofEAE induction in mice injected with PLP-peptide at day 0 and furtherinjected with anti-gp39 antibodies (black bars) or PBS (grey bars) atdays 0, 2, and 6, or at days 4, 6, and 8, or at days 7, 9, and 11,showing that administration of anti-gp39 after induction of the diseasesignificantly prevents EAE.

FIG. 3 is a graphical representation of DAS units measured daily in micetransplanted with donor spleen cells from mice injected with PLP-peptideand anti-gp39 antibodies (black bars) or with donor cells from miceinjected with PLP-peptide alone (grey bars) and injected withPLP-peptide.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to the treatment of T cell mediated autoimmunedisorder such as multiple sclerosis. The disease is treated byadministering an antagonist to a receptor on the surface of T cellswhich mediates contact dependent T cell helper effector function.

As defined herein, a “molecule or receptor which mediates contactdependent helper effector functions” is one which is expressed on a Thcell and interacts with a ligand on an effector cell (e.g., a B cell),wherein the interaction of the receptor with its ligand is necessary forgeneration of an effector cell response (e.g., B cell activation). Inaddition to being involved in effector cell responses, it has been foundthat such a molecule is involved in the response of the T cell toantigen.

In a preferred embodiment, the receptor on the surface of the T cellwhich mediates contact-dependent helper effector functions is gp39. Inthis embodiment, the antagonist is a molecule which inhibits theinteraction of gp39 with its ligand on a cell which presents antigen tothe T cell. A particularly preferred gp39 antagonist is an anti-gp39antibody. Alternatively, the gp39 antagonist is a soluble form of a gp39ligand, for example soluble CD40.

The method of the invention is based at least in part on the observationthat administration of anti-gp39 antibodies to mice prevents inductionof EAE and reverses the disease in animals having EAE. Thus, it has beenfound that an agent which inhibits the interaction of gp39 on a T cellwith its ligands(s) on other cells is effective, both prophylacticallyand therapeutically, in treating a typical T cell mediated autoimmunedisease. This result is surprising in view of previous studies whichhave attributed to gp39 a primary role in regulating B cell responses.The finding that anti-gp39 antibodies are effective in treating T cellmediated autoimmune disease forms the basis of the present invention.According to the invention, subjects having a T cell mediated autoimmunedisease, such as multiple sclerosis, are treated by administration ofagents that mimic the effect of anti-gp39 antibodies.

T Cell Mediated Autoimmune Diseases

The language “autoimmune disorder” is intended to include disorders inwhich the immune system of a subject reacts to autoantigens, such thatsignificant tissue or cell destruction occurs in the subject. The term“autoantigen” is intended to include any antigen of a subject that isrecognized by the immune system of the subject. The terms “autoantigen”and “self-antigen” are used interchangeably herein. The term “self” asused herein is intended to mean any component of a subject and includesmolecules, cells, and organs. Autoantigens may be peptides, nucleicacids, or other biological substances. The language “T cell mediatedautoimmune disorder” is intended to include autoimmune disorders inwhich the reaction to self primarily involves cell-mediated immunemechanisms, as opposed to humoral immune mechanisms. Thus, the methodsof the invention pertain to treatments of autoimmune disorders in whichtissue destruction is primarily mediated through activated T cells andimmune cells other than B lymphocytes. However, even though the methodsof the invention are intended for treatment of autoimmune disorders inwhich reaction to self is primarily mediated by cells other than Bcells, the autoimmune disorders may be characterized by the presence ofautoantibodies. For example, EAE, a T cell mediated autoimmune disorder,which can be treated by a method of the invention, is frequentlyassociated with the presence of autoantibodies to components of thecentral nervous system, such as myelin basic protein. Non limitingexamples of T cell mediated autoimmune disorders that can be treated bythe methods of the invention include multiple sclerosis, EAE, diabetestype I, oophoritis, and thyroiditis.

gp39 Antagonists

According to the methods of the invention, a gp39 antagonist isadministered to a subject to interfere with the interaction of gp39 on Tcells with a gp39 ligand on antigen presenting cells, such as B cellsand thereby to prevent, alleviate or ameliorate the disorder. A gp39antagonist is defined as a molecule which interferes with thisinteraction. As described more fully below, the gp39 antagonist can bean antibody directed against gp39 (e.g., a monoclonal antibody againstgp39), a fragment or derivative of an antibody directed against gp39(e.g., Fab or F(ab)′2 fragments, chimeric antibodies or humanizedantibodies), soluble forms of a gp39 ligand (e.g., soluble CD40),soluble forms of a fusion protein of a gp39 ligand (e.g., solubleCD40Ig), or pharmaceutical agents which disrupt or interfere with thegp39-CD40 interaction.

A. Antibodies

To prepare anti-gp39 antibodies, a mammal (e.g., a mouse, hamster, orrabbit) can be immunized with an immunogenic form of gp39 protein orprotein fragment (e.g., peptide fragment) which elicits an antibodyresponse in the mammal. A cell which expresses gp39 on its surface canalso be used as the immunogen. Alternative immunogens include purifiedgp39 protein or protein fragments. gp39 can be purified from agp39-expressing cell by standard purification techniques, e.g., gp39cDNA (Armitage et al., Nature, 357:80-82 (1992); Lederman et al., J.Exp. Med., 175:1091-1101 (1992); Hollenbaugh et al., EMBO J.,11:4313-4319 (1992)) can be expressed in a host cell, e.g., bacteria ora mammalian cell line, and gp39 protein purified from the cell cultureby standard techniques. gp39 peptides can be synthesized based upon theamino acid sequence of gp39 (disclosed in Armitage et al., Nature,357:80-82 (1992); Lederman et al., J. Exp. Med., 175:1091-1101 (1992);Hollenbaugh et al., EMBO J., 11:4313-4319 (1992)) using known techniques(e.g. F-moc or T-boc chemical synthesis). Techniques for conferringimmunogenicity on a protein include conjugation to carriers or othertechniques well known in the art. For example, the protein can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassay can be used with the immunogen asantigen to assess the levels of antibodies.

Following immunization, antisera can be obtained and, if desired,polyclonal antibodies isolated from the sera. To produce monoclonalantibodies, antibody producing cells (lymphocytes) can be harvested froman immunized animal and fused with myeloma cells by standard somaticcell fusion procedures thus immortalizing these cells and yieldinghybridoma cells. Such techniques are well known in the art. For example,the hybridoma technique originally developed by Kohler and Milstein(Nature (1975) 256:495-497) as well as other techniques such as thehuman B-cell hybridoma technique (Kozbar et al., Immunol. Today (1983)4:72), the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985)(Allen R. Bliss, Inc., pages 77-96), and screening of combinatorialantibody libraries (Huse et al., Science (1989) 246:1275). Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with the protein or peptide and monoclonalantibodies isolated.

The term antibody as used herein is intended to include fragmentsthereof which are specifically reactive with a gp39 protein or peptidethereof or gp39 fusion protein. Antibodies can be fragmented usingconventional techniques and the framents screened for utility in thesame manner as described above for whole antibodies. For example,F(ab′)₂ fragments can be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments. The antibody of the present invention is furtherintended to include bispecific and chimeric molecules having ananti-gp39 portion.

When antibodies produced in non-human subjects are used therapeuticallyin humans, they are recognized to varying degrees as foreign and animmune response may be generated in the patient. One approach forminimizing or eliminating this problem, which is preferable to generalimmunosuppression, is to produce chimeric antibody derivatives, i.e.,antibody molecules that combine a non-human animal variable region and ahuman constant region. Chimeric antibody molecules can include, forexample, the antigen binding domain from an antibody of a mouse, rat, orother species, with human constant regions. A variety of approaches formaking chimeric antibodies have been described and can be used to makechimeric antibodies containing the immunoglobulin variable region whichrecognizes gp39. See, for example, Morrison et al., Proc. Natl. Acad.Sci. U.S.A. 81:6851 (1985); Takeda et al., Nature 314:452 (1985),Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No.4,816,397; Tanaguchi et al., European Patent Publication EP171496;European Patent Publication 0173494, United Kingdom Patent GB 2177096B.It is expected that such chimeric antibodies would be less immunogenicin a human subject than the corresponding non-chimeric antibody.

For human therapeutic purposes the monoclonal or chimeric antibodiesspecifically reactive with a gp39 protein or peptide can be furtherhumanized by producing human variable region chimeras, in which parts ofthe variable regions, especially the conserved framework regions of theantigen-binding domain, are of human origin and only the hypervariableregions are of non-human origin. Such altered immunoglobulin moleculesmay be made by any of several techniques known in the art, (e.g., Tenget al., Proc. Natl. Acad. Sci. USA., 80:7308-7312 (1983); Kozbor et al.,Immunology Today, 4:7279 (1983); Olsson et al., Meth Enzymol., 92:3-16(1982)), and are preferably made according to the teachings of PCTPublication WO92/06193 or EP 0239400. Humanized antibodies can becommercially produced by, for example, Scotgen Limited, 2 Holly Road,Twickenham, Middlesex, Great Britain.

Another method of generating specific antibodies, or antibody fragments,reactive against a gp39 protein or peptide is to screen expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with a gp39 protein or peptide. For example, complete Fabfragments, VH regions and FV regions can be expressed in bacteria usingphage expression libraries. See for example Ward et al., Nature, 341:544-546: (1989); Huse et al., Science, 246: 1275-1281 (1989); andMcCafferty et al., Nature, 348: 552-554 (1990). Screening such librarieswith, for example, a gp39 peptide can identify immunoglobulin fragmentsreactive with gp39. Alternatively, the SCID-hu mouse (available fromGenpharm) can be used to produce antibodies, or fragments thereof.

Methodologies for producing monoclonal antibodies directed against gp39,including human gp39 and mouse gp39, and suitable monoclonal antibodiesfor use in the methods of the invention, are described in PCT PatentApplication No. WO 95/06666 entitled “Anti-gp39 Antibodies and UsesTherefor”, the teachings of which are incorporated by reference.Particularly preferred anti-human gp39 antibodies of the invention aremAbs 24-31 and 89-76, produced respectively by hybridomas 24-31 and89-76. The 89-76 and 24-31 hybridomas, producing the 89-76 and 24-31antibodies, respectively, were deposited under the provisions of theBudapest Treaty with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, on Sep. 2, 1994. The89-76 hybridoma was assigned ATCC Accession Number HB11713 and the 24-31hybridoma was assigned ATCC Accession Number HB 11712.

Recombinant anti-gp39 antibodies, such as chimeric and humanizedantibodies, can be produced by manipulating nucleic acid (e.g., DNA)encoding an anti-gp39 antibody according to standard recombinant DNAtechniques. Accordingly, another aspect of this invention pertains toisolated nucleic acid molecules encoding immunoglobulin heavy or lightchains, or portions thereof, reactive with gp39, particularly humangp39. The immunoglobulin-encoding nucleic acid can encode animmunoglobulin light or heavy chain variable region, with or without alinked heavy or light chain constant region (or portion thereof). Suchnucleic acid can be isolated from a cell (e.g., hybridoma) producing ananti-human gp39 mAb by standard techniques. For example, nucleic acidencoding the 24-31 or 89-76 mAb can be isolated from the 24-31 or 89-76hybridoma, respectively, by cDNA library screening, PCR amplification orother standard technique. Following isolation of, and possible furthermanipulation of, Moreover, nucleic acid encoding an anti-human gp39 mAbcan be incorporated into an expression vector and introduced into a hostcell to facilitate expression and production of recombinant forms ofanti-human gp39 antibodies.

B. Soluble Ligands for gp39

Other gp39 antagonists which can be used to induce T cell tolerance aresoluble forms of a gp39 ligand. A monovalent soluble ligand of gp39,such as soluble CD40 can bind gp39, thereby inhibiting the interactionof gp39 with CD40 on B cells. The term “soluble” indicates that theligand is not permanently associated with a cell membrane. A solublegp39 ligand can be prepared by chemical synthesis, or, preferably byrecombinant DNA techniques, for example by expressing only theextracellular domain (absent the transmembrane and cytoplasmic domains)of the ligand A preferred soluble gp39 ligand is soluble CD40.Alternatively, a soluble gp39 ligand can be in the form of a fusionprotein. Such a fusion protein comprises at least a portion of the gp39ligand attached to a second molecule. For example, CD40 can be expressedas a fusion protein with immunoglobulin (i.e., a CD40Ig fusion protein).In one embodiment, a fusion protein is produced comprising amino acidresidues of an extracellular domain portion of the CD40 molecule joinedto amino acid residues of a sequence corresponding to the hinge, CH2 andCH3 regions of an immunoglobulin heavy chain, e.g., Cγ1, to form aCD40Ig fusion protein (see e.g., Linsley et al. (1991) J. Exp. Med.1783:721-730; Capon et al. (1989) Nature 337, 525-531; and Capon U.S.Pat. No. 5,116,964). The fusion protein can be produced by chemicalsynthesis, or, preferably by recombinant DNA techniques based on thecDNA of CD40 (Stamenkovic et al., EMBO J., 8:1403-1410 (1989)).

An antagonist of the invention is administered to subjects in abiologically compatible form suitable for pharmaceutical administrationin vivo. By “biologically compatible form suitable for administration invivo” is meant a form of the antagonist to be administered in which anytoxic effects are outweighed by the therapeutic effects of the protein.The term subject is intended to include living organisms in which animmune response can be elicited, e.g., mammals. Examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.A gp39 antagonist can be administered in any pharmacological form,optionally in a pharmaceutically acceptable carrier. Administration of atherapeutically active amount of the antagonist is defined as an amounteffective, at dosages and for periods of time necessary to achieve thedesired result. For example, a therapeutically active amount of anantagonist of gp39 may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantagonist to elicit a desired response in the individual. Dosage regimamay be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

The active compound (e.g., antagonist) may be administered in aconvenient manner such as by injection (subcutaneous, intravenous,etc.), oral administration, inhalation, transdermal application, orrectal administration. Depending on the route of administration, theactive compound may be coated in a material to protect the compound fromthe action of enzymes, acids and other natural conditions which mayinactivate the compound. A preferred route of administration is byintravenous injection.

To administer an antagonist of gp39 by other than parenteraladministration, it may be necessary to coat the antagonist with, orco-administer the antagonist with, a material to prevent itsinactivation. For example, an antagonist can be administered to anindividual in an appropriate carrier or diluent, co-administered withenzyme inhibitors or in an appropriate carrier such as liposomes.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Enzyme inhibitors include pancreatic trypsin inhibitor,diisopropyl-fluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water emulsions as well as conventional liposomes(Strejan et al., (1984) J. Neuroimmunol 7:27).

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fingi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating activecompound (e.g., an antagonist of gp39) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient (e.g., antagonist) plusany additional desired ingredient from a previously sterile-filteredsolution thereof.

When the active compound is suitably protected, as described above, theprotein may be orally administered, for example, with an inert diluentor an assimilable edible carrier. As used herein “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the therapeutic compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLE 1 EAE Prevention by Anti-gp39 Antibody Administration

This example demonstrates that administration of anti-gp39 antibodies tomice prevents induction of experimental allergic encephalomyelitis(EAE), an animal model for multiple sclerosis.

EAE is a well characterized model of a T cell mediated autoimmunedisease and is an instructive model for the human autoimmune diseasemultiple sclerosis. EAE can be induced in susceptible animals, such asmice, by immunizing the animals with myelin basic protein (MBP),proteolipid protein (PLP), myelin oliogodendrocyte protein (MOG), orsynthetic peptides based on the sequences of these myelin associatedproteins together with an adjuvant containing pertussis bacteria One totwo weeks after immunization, the animals develop encephalomyelitis,characterized by perivascular infiltrates containing lymphocytes andmacrophages and the development of demyelination in the brain and thespinal cord. The animals show acute, chronic or chronic relapsingparalysis. In this example, the effect of administration of anti-gp39antibodies on development of EAE in susceptible mice was analyzed.

EAE was induced in susceptible mice by subcutaneous injections (day 0)of an emulsion containing 70 μg or 300 μg PLP-peptide in 50 μl PBS and25 μg Mycobacteria tuberculosis (H37RA, Difco) in 50 μl of completeFreuds adjuvant at two sites in the abdominal flanks of the mice. 200 μlBordetella pertussis suspension (10.10¹⁰ in 1 ml PBS) was givenintravenously at the same time as the peptide and two days later. ThePLP-peptide injected in the mice has an amino acid sequencecorresponding to amino acid residues 139 to 151 of rat PLP (Dautigny etal., FEBS Lett. 188:33, 1985). PLP-peptide was synthesized with f-mocprotected aminoacids according to the solid phase synthesis method(Merrifield, J. Am. Chem. Soc. 5:2149, 1963). Immunization with thispeptide results in the development of acute EAE, which is clinically andpathologically identical to that induced by sensitization with wholecentral nervous system (CNS) myelin or with MBP (Tuohy et al., J.Immunol. 142:1523, 1989; Sobel et al., J. Neuropathol. Exp. Neurol.49:468, 1990).

To determine the effect of anti-gp39 on the course of the disease, micewere injected on day 0 with PLP-peptide, as described above, and furtherinjected intraperitoneally with 125 μg hamster anti-gp39 Mabs (Noelle etal., Proc. Natl. Acad Sci. USA 89:6550, 1992) in 200 μl PBS or with 125μg normal hamster antibodies (Serva Feinbiochemica) in 200 μl PBS(control animals) on days 0, 2, and 4. The severity of EAE clinicalsigns was evaluated each day and graded according to average disabilityscale (DAS) scores: grade 0=no clinical signs, grade 1=tail weakness,grade 2=mild paraparesis and ataxia of the hind legs, grade 3=severeparaparesis or ataxia of the hind legs, grade 4=moribund, grade 5=deaddue to EAE.

FIG. 1 represents the course of the disease in mice injected with 70 μgof PLP-peptide (Panel A) or with 300 μg of PLP-peptide (Panel B) andtreated with anti-gp39 or control antibody. DAS scores, which reflectthe severity of the disease, of control mice and anti-gp39 treated miceare depicted in grey and black bars respectively.

The results indicate that animals having received the control antibodydeveloped EAE, whereas animals having received anti-gp39 antibody wereprotected from induction of the disease. For animals having receivedcontrol antibody, the first clinical signs of EAE became apparent on dayeleven. In these animals, the highest DAS score was 2.33, which wasobserved on days 15-22 in animals injected with 75 μg of PLP-peptide(FIG. 1, Panel A, grey bars) and 3.6, observed on days 16-23 in animalsinjected with 300 μg PLP-peptide (FIG. 1, Panel B, grey bars). Incontrast, animals which received the anti-gp39 monoclonal antibodiesshowed no clinical signs after induction of EAE with 75 μg ofPLP-peptide (FIG. 1, Panel A, black bars) and only minor clinical signs,which completely disappeared at day 31, after induction of the diseasewith 300 μg of PLP-peptide (FIG. 1, Panel B, black bars).

Thus, administration of anti-gp39 antibodies to mice completelyinhibited induction of EAE in these mice.

Induction of EAE by adoptive transfer of myelin reactive T-cellsisolated from animals immunized with myelin components or obtained afterin vitro activation with myelin components indicates that especiallyactivated T-cells are responsible for the development of clinicalcharacteristics after the inductive phase (Pettinelli and McFarlin, J.Immunol. 127:1420, 1979; Mokhtarion et al., Nature 309:356, 1984; Veenet al., J. Neuroimmunol. 21:183, 1989). However, the experimentsdescribed herein show that administration of anti-gp39 monoclonalantibodies prevents the development of EAE. In control groups,significant anti-PLP-peptide antibody responses were observed on day 14(absorbance 1.92) and 21 (absorbance 2.15) in animals in which EAE wasinduced with low and high PLP-peptide dose, respectively. In contrast,significant anti-PLP-peptide antibody responses in the gp39 treatedanimals were observed first on day 14, and reached plateau levels on day31 (absorbance 0.928) and 40 (absorbance 1.54) in animals which wereinjected with low and high PLP-peptide dose, respectively. Thegeneration of significant anti-PLP-peptide antibody responses inanti-gp39 monoclonal antibodies treated mice, was postponed until day14, which indicates that the anti-gp39 monoclonal antibodies had someeffect on antibody production.

Thus, this example demonstrates that anti-gp39 prevent development ofEAE and indicate that anti-gp39 antibodies can be used for treating Tcell mediated autoimmune diseases, such as multiple sclerosis.

EXAMPLE 2 Reversal of EAE by Anti-gp39 Antibody Administration

Example 1 showed the inhibitory effect of anti-gp39 antibody oninduction of EAE. Thus, it was demonstrated that immunization of themice at the time of induction of the disease prevented development ofthe disease. This example shows that administration of the antibodyafter induction of the disease leads to regression of the disease.

In this example, EAE was induced in female SJL/j mice (10-12 weeks old)by injection of an emulsion containing 150 μg PLP-peptide prepared asdescribed above. For determining the effect of the anti-gp39 antibodieswhen administered to the mice after induction of the disease, mice wereinjected intraperitoneally with 125 μg anti-gp39 monoclonal antibodies(Noelle et al., Proc. Natl. Acad. Sci. USA 89:6550, 1992) in 200 μl ofPBS (anti-gp39 treated mice) or with 200 μl PBS alone (control mice) ondays 0, 2 and 4, on days 4, 6 and 8, or on days 7, 9 and 11. The resultsare presented as percentage suppression, and are a comparison of thetotal of daily DAS scores (from day 12 to 28) in anti-gp39 treatedanimals and in control animals.

The results, which are presented in FIG. 2, indicate that administrationof the first dose of anti-gp39 antibodies as late as 7 days afterinjection into the mice of PLP-peptide results in more than 60%suppression of the disease. Thus, anti-gp39 antibodies are capable ofreversing or suppressing EAE.

The results further indicate that even though administration of a firstdose of antibody only 7 days after induction of the disease in miceresults in significant suppression of development of the disease,anti-gp39 treatment is somewhat more efficient when the first dose ofantibody is administered sooner after induction of the disease.

Thus, administration of anti-gp39 antibodies to mice protects these micefrom developing EAE upon induction of the disease and suppresses thedisease in mice having EAE.

EXAMPLE 3 Suppression of EAE After Spleen Cell Transfer of gp39 TreatedMice

Regulatory suppessor T-cells have been detected in Lewis rat, which havebeen recovered from EAE (Pesoa et al., J. Neuroimmunol. 7:131, 1984) andafter oral administration of myelin components (Lider et al., J.Immunol. 142:748, 1989; Hafler et al., Ann. NY Acad. Sci. 636:251,1991). It was postulated by Karpus and Swanborg (J. Immunol. 143:3492,1989) that CD4⁺ suppressor T-cells isolated from rats which haverecovered from EAE, can down regulate EAE T-effector cells bydifferential inhibition of lymphokine production. In contrast,suppression of EAE in Lewis rats by oral administration of MBP ismediated by CD8⁺ T-cells (Miller et al., J. Exp. Med 174:791, 1991). Todetermine whether T cells of mice having been protected from EAE byadministration of anti-gp39 antibody, are capable of protecting naiveanimals from EAE, the following example was undertaken.

In this example, a first set of mice were injected with 150 μg ofPLP-peptide and a second set of mice were injected with 150 μg ofPLP-peptide and anti-gp39 antibody according to the protocol describedin Example 1. Four months later the mice were sacrified by CO₂euthanasia and the spleens were removed. Erythrocytes were removed bystandard ammoniumchloride treatment (Mishell and Shiigi, SelectedMethods in Cellular Immunology, W. H. Freeman and Company, 1980). Cellsof individual spleens (500 μl) were injected i.v. in naive 5 Gyirradiated recipient female SLJ/j mice (10-12 weeks old). Two days aftercell transfer, mice were challenged by intraperitoneal injection with150 μg PLP-peptide following the procedure described in Example 1 andDAS scores determined.

FIG. 3 represents the DAS scores of the animals. The results demonstratethat mice have been transplanted with spleen cells from animalsinitially injected with PLP-peptide and anti-gp39 antibodies areprotected from development of the disease, whereas mice transplantedwith spleen cells from animals initially injected with PLP-peptide onlydevelop EAE. Moreover, considering that the estimated half-life ofantibodies is 12 days, it may be expected that no antibodies werepresent in the spleen cells transplanted in the mice. Therefore, theprotective effect conferred by donor spleen cells from animals havingreceived PLP-peptide and anti-gp39 antibodies cannot be explained by thepresence of anti-gp39 antibodies. DAS scores of these mice indicatesthat suppression of EAE in the recipient mice is most likely due to thepresence of a T-suppressor cell population in the transferred spleencell suspension and that this T-suppressor cell population overrules theT-effector cell population efficiently.

EXAMPLE 4 Detection of gp39 Positive Th-cells

This example demonstrates the presence of gp39 positive cells in thecentral nervous system of human subjects having multiple sclerosis.

Human autopsy central nervous system (CNS) tissues were obtained fromthe Netherlands brain bank, Amsterdam, the Netherlands. Gp39 positivecells were detected with an CD40-Ig fusion protein according to methodsknown in the art. CNS tissue sections of an MS patient and an Alzheimerpatient were stained with CD40-Ig. In this example, only CNS tissues ofMS-patients in which anti-MBP antibody forming cells were detectedpreviously were used. The results of the staining show the presence ofgp39 positive cells in a 8 μm coronal cerebrum section of an MS patient,but no gp39 positive cells were detected in coronal cerebrum sections ofAlzheimer patients. Thus, gp39 positive cells were detected only in CNStissue sections of MS patients. The presence of gp39 positive cells inMS-patient CNS tissue together with the detection of anti-MBP antibodyforming cells in CNS tissues of MS-patients only and not in control CNStissues indicates that such cells play an role in the pathologicalaffected CNS tissues of MS patients.

In this study we have shown that suppression of B-cell activation by theadministration of anti-gp39 Mabs, can result in the complete preventionof EAE development, dependent on the dose of antigen by which EAE wasinduced and dependent on the time period between EAE induction andadministration of anti-gp39 Mabs. Although the exact mechanismsresponsible for EAE induction and development are not clear, these dataindicate that anti-gp39 antibody can be used for treatment of autoimmunediseases.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for preventing or inhibiting thesymptoms of multiple sclerosis in a human subject comprisingadministering to said human subject a prophylactially effective amountof a gp39 antagonist, wherein said gp39 antagonist is an antibody orfragment thereof that specifically binds gp39 (CD40 ligand).
 2. Themethod of claim 1, wherein said anti-gp39 antibody is a monoclonalantibody.
 3. The method of claim 2, wherein said antibody is ananti-human gp39 antibody.
 4. The method of claim 3, wherein saidantibody is selected from the group consisting of the monoclonalantibody produced by the 89-76 hybridoma, ATCC Accession Number HB 11713and the 24-31 hybridoma, ATCC Accession Number HB
 11712. 5. The methodof claim 3, wherein said antibody is a monoclonal antibody containingconstant regions and variable regions derived from antibodies ofdifferent species.
 6. The method of claim 3, wherein said antibody is ahumanized monoclonal antibody.